Iron is an essential element but is toxic in excess. Malregulation of iron transport results in tissue injury, either from iron deprivation or overload. The concentration of free iron within cells is determined by regulated transmembrane iron import and export and by iron storage. We propose to identify and characterize systems that affect the transport of iron across the plasma membrane and into the vesicular apparatus. We will continue our approach of using yeast genetics to identify genes involved in iron metabolism. We have shown that the high affinity iron transport system of yeast involves a multicopper oxidase Fet3 and a transmembrane permease Ftr1. Transcription of this transport system is heme dependent and in the absence of heme transcription is repressed. We will determine how the repressor senses heme. Yeast and plants store iron in the vacuole. We identified the iron transporter Ccc1 that mediates iron export from cytosol to vacuole. We have shown that transcription of CCC1 is increased by iron. We will determine how the transcriptional activator of vacuolar iron import, Yap5, senses iron. Vertebrates store iron in ferritin and ferritin accumulation responds to increased cytosolic iron. We determined that expression of the iron exporter ferroportin or treatment with the permeable iron chelator desferasirox leads to ferritin iron release followed by ferritin degradation by the proteasome. We will determine biochemical changes in ferritin following iron release, how ferritin is marked for degradation and how ferritin is disassembled. The concentration of serum ferritin is a useful marker of iron overload disease, as there is a strong correlation with hepatic iron stores in hereditary hemochromatosis. We have determined the conditions that lead to ferritin secretion. Our data show that ferritin monomers can gain access to the secretory apparatus of cells. We propose to identify the sequences in ferritin that permit secretion. We will also test the hypothesis that secretion of ferritin is a mechanism that protects cells from ferritin induced decrease in free cytosolic iron.